As the primary controllers of epithelium regeneration, intestinal stem cells (ISCs) at the bottom of the crypt must maintain a balance between self-renewal and differentiation. However, it is still unclear how the stem cells maintain tissue homeostasis in response to daily variations in cell numbers or after injury. It is also well known that the ability to repair damage is reduced in aging, but it is not known what mechanism(s) underly this process. We have recently developed a chronic preparation for in vivo imaging with multiphoton microscopy which allows the monitoring the ISC niche in real-time in mice expressing green fluorescent protein in Lgr5+ ISCs. The goal is to directly track and identify how stem cells maintain their balance in the intestinal crypts. Next generation multiphoton microscopy with an in vivo imaging preparation with femtosecond laser ablation is used to ablate individual cells of a specific type to perturb the crypt. Time lapse imaging captures changes in cell number, position, motion and marker expression to identify how the various populations of stem cell respond. Upon ablation, the targeted cells lost their shape and moved out of the plane of the crypt base towards the intestinal lumen. Immediately adjacent cells appeared to move into the space left by the ablated cell, suggesting that the niche cells actively move around in response to the pattern disruption. This proposal tests the hypothesis that age and underlying pathology reduces the efficacy of these newly discovered dynamics, which can be rescued by age-delaying agents. The expectation is that these motions are involved in protecting the stem cells from damaging factors spilling from injured cells. The new optical tools have identified a potential new function of ISCs. In addition to generating daughter cells to replenish the epithelium, Lgr5+ ISCs appear to be mechanically active in eliminating damaged cells. This adds a new function to the repertoire of ISC actions. Collectively, the results suggest that there is an active process that involves cell migration in addition to cell division for maintaining homeostasis in the intestinal crypt and epithelium. !